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1.
Gene ; 764: 145062, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32860900

RESUMO

Recently, DNA-based methods have proved to be accurate, fast and sensitive for meat authentication. According to the European Union, the food safety standards require accurate and detailed composition information of the meat products. Therefore, an accurate, fast and cost-effective identification methodology is needed. In this study, multiplex PCR coupled with 12S rDNA sequencing was employed for the detection of meat adulteration in two red meat products (frozen beef liver and cold cut samples, respectively) in Egypt. Multiplex PCR allowed the identification of ruminant, poultry, pork, and donkey residuals in processed red meat products (cold cuts) in a single step PCR reaction. Preliminary uniplex PCR was performed to evaluate primers specificity using DNA extracted from the positive control samples. The primers produced specific fragments for ruminant, poultry, pork, and donkey as follows: 271, 183, 531 and 145 bp, respectively. Multiplex PCR revealed that none of the samples was contaminated by porcine or donkey residuals, but 62.5% of all tested processed beef samples contained poultry contaminants. The sensitivity of this method was 0.01 ng/µL for beef, poultry and donkey and 0.1 ng/µL for pig. Another promising finding is the identification of all frozen beef liver samples as a cattle species (Bos taurus) through PCR-sequencing of a short fragment of 12S rRNA gene. Finally, we recommend the employment of multiplex PCR and PCR-sequencing of 12S rDNA for quality control in routine analysis of processed and frozen meat products.


Assuntos
Contaminação de Alimentos/análise , Indústria Alimentícia/normas , Produtos da Carne/análise , Reação em Cadeia da Polimerase Multiplex , RNA Ribossômico/genética , Animais , Bovinos/genética , Galinhas/genética , Egito , Limite de Detecção , Produtos da Carne/normas , Carne Vermelha/análise , Carne Vermelha/normas , Análise de Sequência de DNA/métodos , Especificidade da Espécie
2.
BMC Infect Dis ; 20(1): 752, 2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-33054726

RESUMO

BACKGROUND: Molecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients. METHODS: In this study 381 M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n = 131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene. RESULTS: The sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p = 0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p = < 0.0001, < 0.0001 & 0.0014 respectively) was also found. CONCLUSIONS: This rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains.


Assuntos
Proteínas de Bactérias/genética , Catalase/genética , RNA Polimerases Dirigidas por DNA/genética , Mycobacterium tuberculosis/genética , Pentosiltransferases/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antituberculosos/farmacologia , Criança , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Genótipo , Humanos , Índia/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex/métodos , Taxa de Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Adulto Jovem
3.
PLoS Biol ; 18(10): e3000867, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33027248

RESUMO

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Reação em Cadeia da Polimerase Multiplex/normas , Pneumonia Viral/diagnóstico , RNA Viral/genética , Kit de Reagentes para Diagnóstico/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Betacoronavirus/patogenicidade , Estudos de Casos e Controles , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/virologia , Primers do DNA/normas , Células HEK293 , Humanos , Limite de Detecção , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , Estados Unidos
4.
Sci Rep ; 10(1): 18764, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127953

RESUMO

Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.


Assuntos
Técnicas de Laboratório Clínico/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase Multiplex/normas , Reprodutibilidade dos Testes , Mucosa Respiratória/virologia
5.
Artigo em Inglês | MEDLINE | ID: mdl-33053146

RESUMO

Meningoencephalitis is a syndrome of multiple etiologies associated with important morbidity and mortality. It may be caused by various infectious agents (viruses, bacteria, parasites and fungi). Establishing the etiology of meningoencephalitis is crucial for early and specific treatment. Molecular assays such as the multiplex polymerase chain reaction (PCR) offer an alternative in diagnosing central nervous system infections. This study aimed to describe the performance of an automated multiplex molecular test from patients with suspected meningitis and meningoencephalitis in a tertiary referral complex in Medellin, Colombia. Thus, a prospective study was performed in 638 cerebrospinal fluid samples from January 2017 to July 2019. Molecular detections were carried out by means of the FilmArray® Meningitis/Encephalitis (M/E) Panel from bioMérieux, France, and by conventional tests. Univariate analyses for microbiological and demographic characteristics were performed. Accuracy of the bacterial/fungal PCR assay compared to cultures was also performed. Among patients, 57.7% were male, the median age was 24 (IQR: 6 - 47) years old. The overall positivity was 15.2% (97 detections) and viruses were detected in 45.5% of the samples, bacteria in 43.5% and fungi in 10.8%. The most frequent etiological agents were: Streptococcus pneumoniae (16%), Cryptococcus neoformans/gatti (11.3%) and Herpes simplex virus (10.3%). Four double detections were found. Almost half of positive detections were in patients under 15 years old. This molecular approach is reliable and easily implantable into a laboratory routine, increasing the capacity of detection of bacterial and viral causative agents of meningitis, possibly playing a relevant role in the clinical context.


Assuntos
Meningite/epidemiologia , Meningoencefalite/epidemiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Adolescente , Adulto , Criança , Colômbia/epidemiologia , Feminino , Humanos , Masculino , Meningite/etiologia , Meningoencefalite/etiologia , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
6.
BMC Infect Dis ; 20(1): 750, 2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-33050903

RESUMO

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC). Mapping the genetic diversity of MTBC in high TB burden country like Ethiopia is important to understand principles of the disease transmission and to strengthen the regional TB control program. The aim of this study was to investigate the genetic diversity of Mycobacterium tuberculosis complex (MTBC) isolates circulating in the South Omo, southern Ethiopia. METHODS: MTBC isolates (N = 156) were genetically analyzed using spacer oligotyping (spoligotyping) and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing. Major lineages and lineages were identified using MTBC databases. Logistic regression was used to correlate patient characteristics with strain clustering. RESULTS: The study identified Euro-American (EA), East-African-Indian (EAI), Indo-Oceanic (IO), Lineage_7/Aethiops vertus, Mycobacterium bovis and Mycobacterium africanum major lineages in proportions of 67.3% (105/156), 22.4% (35/156), 6.4% (10/156), 1.9% (3/156), 1.3% (2/156) and 0.6% (1/156), respectively. Lineages identified were Delhi/CAS 23.9% (37/155), Ethiopia_2 20.6% (32/155), Haarlem 14.2% (22/155), URAL 14.2%(22/155), Ethiopia_3 8.4% (13/155), TUR 6.5% (10/155), Lineage_7/Aethiops vertus 1.9% (3/155), Bovis 1.3% (2/155), LAM 1.3% (2/155), EAI 0.6% (1/155), X 0.6% (1/155) and Ethiopia H37Rv-like strain 0.6% (1/155). Of the genotyped isolates 5.8% (9/155) remained unassigned. The recent transmission index (RTI) was 3.9%. Orphan strains compared to shared types (AOR: 0.09, 95% CI: 0.04-0.25) were associated with reduced odds of clustering. The dominant TB lineage in pastoral areas was EAI and in non-pastoral areas was EA. CONCLUSION: The epidemiological data, highly diverse MTBC strains and a low RTI in South Omo, provide information contributing to the TB Control Program of the country.


Assuntos
Variação Genética , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Etiópia/epidemiologia , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Repetições Minissatélites/genética , Epidemiologia Molecular , Reação em Cadeia da Polimerase Multiplex , Mycobacterium bovis/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose Pulmonar/microbiologia , Adulto Jovem
7.
BMC Infect Dis ; 20(1): 741, 2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-33036564

RESUMO

BACKGROUND: Cholera remains a major global health challenge. Uvira, in the Democratic Republic of the Congo (DRC), has had endemic cholera since the 1970's and has been implicated as a possible point of origin for national outbreaks. A previous study among this population, reported a case confirmation rate of 40% by rapid diagnostic test (RDT) among patients at the Uvira Cholera Treatment Centre (CTC). This study considers the prevalence and diversity of 15 enteric pathogens in suspected cholera cases seeking treatment at the Uvira CTC. METHODS: We used the Luminex xTAG® multiplex PCR to test for 15 enteric pathogens, including toxigenic strains of V. cholerae in rectal swabs preserved on Whatman FTA Elute cards. Results were interpreted on MAGPIX® and analyzed on the xTAG® Data Analysis Software. Prevalence of enteric pathogens were calculated and pathogen diversity was modelled with a Poisson regression. RESULTS: Among 269 enrolled CTC patients, PCR detected the presence of toxigenic Vibrio cholerae in 38% (103/269) of the patients, which were considered to be cholera cases. These strains were detected as the sole pathogen in 36% (37/103) of these cases. Almost half (45%) of all study participants carried multiple enteric pathogens (two or more). Enterotoxigenic Escherichia coli (36%) and Cryptosporidium (28%) were the other most common pathogens identified amongst all participants. No pathogen was detected in 16.4% of study participants. Mean number of pathogens was highest amongst boys and girls aged 1-15 years and lowest in women aged 16-81 years. Ninety-three percent of toxigenic V. cholerae strains detected by PCR were found in patients having tested positive for V. cholerae O1 by RDT. CONCLUSIONS: Our study supports previous results from DRC and other cholera endemic areas in sub-Sahara Africa with less than half of CTC admissions positive for cholera by PCR. More research is required to determine the causes of severe acute diarrhea in these low-resource, endemic areas to optimize treatment measures. TRIAL REGISTRATION: This study is part of the impact evaluation study entitled: "Impact Evaluation of Urban Water Supply Improvements on Cholera and Other Diarrheal Diseases in Uvira, Democratic Republic of Congo" registered on 10 October 2016 at clinicaltrials.gov Identification number: NCT02928341 .


Assuntos
Cólera/epidemiologia , Criptosporidiose/epidemiologia , Cryptosporidium/genética , Diarreia/epidemiologia , Surtos de Doenças , Escherichia coli Enterotoxigênica/genética , Infecções por Escherichia coli/epidemiologia , Vibrio cholerae/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Pré-Escolar , Cólera/microbiologia , Criptosporidiose/parasitologia , República Democrática do Congo/epidemiologia , Testes Diagnósticos de Rotina , Diarreia/microbiologia , Doenças Endêmicas , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Prevalência , Microbiologia da Água , Adulto Jovem
8.
Wei Sheng Yan Jiu ; 49(5): 823-858, 2020 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-33070830

RESUMO

OBJECTIVE: Multiplex real-time PCR for the identification of 15 Salmonella serovars was developed. METHODS: Through the Salmonella genome comparison, 12 membrane proteins STM4497 gene can be used to identify 15 Salmonella serovars, and these 12 genes were respectively listed as A-L genes. Then primers were designed according to A-L gene conserved sequences, and then multiplex real-time PCR was established assessed with the evaluation of the limit detection, sensitivity, specificity, and repeatability. The 206 Salmonella strains were identified using multiplex real-time PCR with the comparison of the serum slide agglutination assay. RESULTS: The limit detection of multiplex PCR ranged from 1. 1×10~(-3)-1. 2×10~(-3) ng/µL. The target genes were 100% specificity, and the relative standard deviation was lower than 2. 97%. Compared with the serum slide agglutination assay, Kappa ranged 0. 92-1. 00. CONCLUSION: The multiplex real-time PCR can be used to identify 15 Salmonella serovars, which is rapid, accurate and specific.


Assuntos
Infecções por Salmonella , Salmonella , Humanos , Reação em Cadeia da Polimerase Multiplex , Reação em Cadeia da Polimerase em Tempo Real , Salmonella/genética , Infecções por Salmonella/diagnóstico , Sorogrupo
9.
Viruses ; 12(10)2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33066701

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , DNA Complementar/análise , DNA Complementar/genética , DNA Viral/análise , DNA Viral/genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Epidemiologia Molecular , Reação em Cadeia da Polimerase Multiplex , Pandemias , Pneumonia Viral/diagnóstico , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência
10.
Biomed Res Int ; 2020: 7610678, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33029522

RESUMO

Background: There is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and a surge of SARS-CoV-2 cases, especially in limited-resource settings. Therefore, the combination of an optimal assay kit is necessary. Methods: We compared the ability to screen SARS-CoV-2 among three primer-probe sets in two different master mixes, Invitrogen™ SuperScript™ III One-Step RT-PCR and LightCycler Multiplex RNA Virus Master. Results: The assay with TIB-Molbiol, IDT, and Phu Sa sets for LightCycler Multiplex RNA Virus Master or Invitrogen™ SuperScript™ III One-Step RT-PCR showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. R squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. Conclusions: Our findings indicated that TIB-Molbiol, IDT, and Phu Sa primer-probe sets could be beneficial for the laboratory screening of SARS-CoV-2 by RT-qPCR assay of E gene. There is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for COVID-19.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , Pneumonia Viral/diagnóstico , Sondas RNA/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade
11.
Int J Food Microbiol ; 335: 108885, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-32947145

RESUMO

Worldwide, Listeria monocytogenes is a common foodborne pathogen and serotyping is necessary for traceability and surveillance. Due to high accuracy and speed, PCR is commonly used for serotyping by targeting specific genes, such as lmo1118 and ORF2110 for 1/2c-3c and 4b-4d-4e, respectively. Single nucleotide polymorphisms (SNPs) are single base alterations at specific loci which are associated with various phenomena. In this study, a method was explored to mine specific SNPs from 253 L. monocytogenes genomes with known serotypes by writing Python programming language script. After blasting all the CDS, seventeen candidate genes with specific SNPs were obtained and these genes contained multiple types of SNPs, including lineages I, II, III, 1/2a-3a, 1/2b-3b-7, and 1/2c-3c specific SNPs. The sensitivity and specificity of these candidate SNP sites are higher than 85% in the genomes examined. Combining lineage I-specific, lineage II-specific, 1/2b-3b-7-specific, and 1/2c-3c-specific SNP sites together, using allele-specific multiplex PCR, we could serotype major L. monocytogenes serotypes. This method was used for 60 L. monocytogenes strains isolated from various food samples and the serotyping results were 100% identical to those of the currently accepted method with the reduced primers from ten to eight. Our results indicate that allele-specific multiplex PCR after mining specific SNPs from common genome database can be used for bacterial typing.


Assuntos
Microbiologia de Alimentos/métodos , Listeria monocytogenes/isolamento & purificação , Listeriose/diagnóstico , Polimorfismo de Nucleotídeo Único/genética , Sorotipagem/métodos , Alelos , Humanos , Listeria monocytogenes/genética , Reação em Cadeia da Polimerase Multiplex , Sensibilidade e Especificidade , Sorogrupo
12.
PLoS One ; 15(9): e0239467, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970731

RESUMO

Dystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection of individual transcript molecules at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We instead multiplex within a single transcript, using probes targeted to the 5' and 3' regions of muscle dystrophin mRNA. Our approach shows this method can reveal transcriptional dynamics in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs in quantitative fashion (with the latter absent in dystrophic muscle, yet restored following therapeutic intervention). We show that even in healthy muscle, immature dystrophin mRNA predominates (60-80% of total), with the surprising implication that the half-life of a mature transcript is markedly shorter than the time invested in transcription: at the transcript level, supply may exceed demand. Our findings provide unique spatiotemporal insight into the behaviour of this long transcript (with implications for therapeutic approaches), and further suggest this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.


Assuntos
Distrofina/genética , Expressão Gênica/genética , Hibridização In Situ/métodos , Animais , Distrofina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Multiplex/métodos , Músculos/metabolismo , RNA Mensageiro/genética , Transcrição Genética/genética
13.
Bull Cancer ; 107(11): 1091-1097, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980144

RESUMO

PURPOSE: This study aims to analyze sensitivities and polymorphisms of the Bethesda panel markers (BAT25, BAT26, D2S123, D5S346 and D17S250) for microsatellite instability testing in Chinese from Jiangsu Province and their clinical implication. METHODS: MSI, sensitivity and polymorphism analysis in 541 colorectal cancer (CRC) patients were detected by fragment analysis. RESULTS: Five hundred and twenty-five tissue samples and 541 blood samples of the 541 sample pairs were successfully amplified. Thirty-four (6.5%) cases were MSI-high (MSI-H) while 33 (6.3%) and 458 (87.2%) were MSI-low (MSI-L) and microsatellite stable (MSS), respectively. BAT26 (85.3%) exhibited the highest instability followed by BAT25 (82.4%), D2S123 (67.6%), D17S250 (64.7%) and D5S346 (50.0%) in MSI-H cases. The median ages of CRC patients with LS, MSI-H, MSI-L and MSS status were 38-43, 48, 60 and 63, respectively. 75.0%, 44.1%, 12.1% and 7.0% CRC cases were mucinous carcinomas in LS, MSI-H, MSI-L and MSS group, respectively. For D2S123, D17S250 and D5S346, heterozygosity was 80.8%, 74.1% and 57.7% and sizes of polymorphic variation range (PVR) were 207bp to 234bp, 140bp to 169bp and 109bp to 137bp, respectively. For D2S123 and D5S346, there was a bimodal distribution distinguishing the D17S250 from an indistinct trimodal or tetramodal distribution. CONCLUSION: MSI-H cases showed earlier onset and higher proportion of mucinous carcinomas. Mononucleotide BAT26 and BAT25 exhibited higher sensitivity than dinucleotides D2S123, D17S250 and D5S346 in the Chinese population. The dinucleotide markers were highly polymorphic with high percent of heterozygosity, great variation in repeat length and non-normal distribution in Chinese population from Jiangsu Province.


Assuntos
Adenocarcinoma Mucinoso/genética , Neoplasias Colorretais/genética , Marcadores Genéticos , Instabilidade de Microssatélites , Polimorfismo Genético , Adulto , China , Neoplasias Colorretais/etnologia , Reparo de Erro de Pareamento de DNA , Primers do DNA , Feminino , Heterozigoto , Humanos , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex/métodos , Técnicas de Amplificação de Ácido Nucleico
14.
PLoS One ; 15(9): e0239403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946527

RESUMO

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.


Assuntos
Betacoronavirus/genética , Primers do DNA/normas , Genoma Viral/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Sequenciamento Completo do Genoma/métodos , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Primers do DNA/metabolismo , Dimerização , Amplificação de Genes , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Carga Viral
15.
BMC Infect Dis ; 20(1): 679, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948121

RESUMO

BACKGROUND: Since December 2019, the coronavirus disease 2019 (COVID-19) has infected more than 12,322,000 people and killed over 556,000 people worldwide. However, Differential diagnosis remains difficult for suspected cases of COVID-19 and need to be improved to reduce misdiagnosis. METHODS: Sixty-eight cases of suspected COVID-19 treated in Wenzhou Central Hospital from January 21 to February 20, 2020 were divided into confirmed and COVID-19-negative groups based on the results of real-time reverse transcriptase polymerase chain reaction (RT-PCR) nucleic acid testing of the novel coronavirus in throat swab specimens to compare the clinical symptoms and laboratory and imaging results between the groups. RESULTS: Among suspected patients, 17 were confirmed to COVID-19-positive group and 51 were distinguished to COVID-19-negative group. Patients with reduced white blood cell (WBC) count were more common in the COVID-19-positive group than in the COVID-19-negative group (29.4% vs 3.9%, P = 0.003). Subsequently, correlation analysis indicated that there was a significant inverse correlation existed between WBC count and temperature in the COVID-19-positive patients (r = - 0.587, P = 0.003), instead of the COVID-19-negative group. But reduced lymphocyte count was no different between the two groups (47.1% vs 25.5%, P = 0.096). More common chest imaging characteristics of the confirmed COVID-19 cases by high-resolution computed tomography (HRCT) included ground-glass opacities (GGOs), multiple patchy shadows, and consolidation with bilateral involvement than COVID-19-negative group (82.4% vs 31.4%, P = 0.0002; 41.2% vs 17.6% vs P = 0.048; 76.5% vs 43.1%, P = 0.017; respectively). The rate of clustered infection was higher in COVID-19-positive group than COVID-19-negative group (64.7% vs 7.8%, P = 0.001). Through multiplex PCR nucleic acid testing, 2 cases of influenza A, 3 cases of influenza B, 2 cases of adenovirus, 2 cases of Chlamydia pneumonia, and 7 cases of Mycoplasma pneumoniae were diagnosed in the COVID-19-negative group. CONCLUSIONS: WBC count inversely correlated with the severity of fever, GGOs, multiple patchy shadows, and consolidation in chest HRCT and clustered infection are common but not specific features in the confirmed COVID-19 group. Multiplex PCR nucleic acid testing helped differential diagnosis for suspected COVID-19 cases.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Adulto , China/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Diagnóstico Diferencial , Feminino , Febre/diagnóstico , Humanos , Influenza Humana/diagnóstico , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Radiografia Torácica , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Tomografia Computadorizada por Raios X
16.
PLoS One ; 15(9): e0239258, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32961548

RESUMO

BACKGROUND: Viral infections in children and adolescents with malignancy are commonly encountered and have a significant impact on morbidity and mortality. Studies and epidemiological data regarding viral infections in children with cancer in developing countries are lacking. This retrospective cohort study aims to assess the burden of viral infections in children and adolescents with cancer, by assessing prevalence, risk factors, as well as morbidity and mortality of common viruses over a period of 8 years. METHODS AND FINDINGS: Medical records of cancer patients treated at the Children Cancer Center of Lebanon were reviewed and 155 participants under the age of 21 were identified with at least one documented viral infection during the period from July 2009 to November 2017. This subset included 136 participants with active malignancy and 19 participants with a history of cancer who underwent hematopoietic stem cell transplantation [HSCT] and were in remission; the latter group was analyzed separately. Information regarding participant characteristics, hospital course, and complications were obtained. Associations between viral infections and certain factors were assessed. In the cohort, 64% were male, 81% were Lebanese. In participants with active malignancy, 90% received chemotherapy in the 6 months preceding the viral infection episode, 11% received radiotherapy. 51% of participants were neutropenic at the time of viral detection, and 77% were lymphopenic. 17% experienced a bacterial co-infection, and 3 experienced a viral co-infection. Among 162 viral infection episodes, clinically diagnosed skin infections, mainly herpes simplex virus type 1 and varicella-zoster virus, were the most common [44% of cases]. These were followed by laboratory-proven systemic herpes infections: cytomegalovirus [14%] and Epstein-Barr virus [6%]. Respiratory viruses: influenza and respiratory syncytial virus, accounted for 9% and 4%, respectively, whereas rotavirus represented 11% and BK virus represented 3% of cases. Acute lymphocytic leukemia was the most prevalent neoplasia [57%]. Fever was the most common presenting symptom [55%] and febrile neutropenia was the reason for admission in 24% of cases. The mean length of stay was significantly longer in participants with cytomegalovirus infections and significantly lower in rotavirus infection. Admission to the ICU occurred in 9%, complications in 8%, and mortality in 5%. Participants with viral infections post-HSCT were noted to have a significantly longer length of hospital stay compared to non-HSCT participants, with no other significant differences in clinical course and outcome. The study was limited by its retrospective nature and by the late introduction and underuse of multiplex PCR panels, which may have led to underdiagnosis of viral infections. CONCLUSIONS: Viral infections were prevalent in our sample of cancer patients and may have contributed to morbidity and mortality. Newly available viral diagnostics are likely to vastly increase the number and scope of detectable viral infections in this population. Prospective studies using multiplex PCR technology with systematic testing of patients will be more helpful in defining the burden of viral infections. Furthermore, efforts at antimicrobial stewardship would benefit from the identification of viral causes of infection and limit the unnecessary use of antibiotics in the pediatric cancer population.


Assuntos
Infecções por Citomegalovirus/epidemiologia , Influenza Humana/epidemiologia , Neoplasias/epidemiologia , Infecções Respiratórias/epidemiologia , Adolescente , Criança , Criança Hospitalizada , Pré-Escolar , Coinfecção/complicações , Coinfecção/diagnóstico , Coinfecção/virologia , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/virologia , Feminino , Transplante de Células-Tronco Hematopoéticas , Hospitais , Humanos , Lactente , Influenza Humana/complicações , Influenza Humana/diagnóstico , Influenza Humana/virologia , Líbano/epidemiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Neoplasias/complicações , Neoplasias/diagnóstico , Neoplasias/virologia , Pediatria , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Vírus Sincicial Respiratório Humano/patogenicidade , Infecções Respiratórias/complicações , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Estudos Retrospectivos , Infecções por Rotavirus/complicações , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/patologia
17.
Epidemiol Infect ; 148: e206, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32867880

RESUMO

Enterotoxigenic Escherichia coli (ETEC) is a well-established cause of traveller's diarrhoea and occasional domestic foodborne illness outbreaks in the USA. Although ETEC are not detected by conventional stool culture methods used in clinical laboratories, syndromic culture-independent diagnostic tests (CIDTs) capable of detecting ETEC have become increasingly prevalent in the last decade. This study describes the epidemiology of ETEC infections reported to the Minnesota Department of Health (MDH) during 2016-2017. ETEC-positive stool specimens were submitted to MDH to confirm the presence of ETEC DNA by polymerase chain reaction (PCR). Cases were interviewed to ascertain illness and exposures. Contemporaneous Salmonella cases were used as a comparison group in a case-case comparison analysis of risk factors. Of 222 ETEC-positive specimens received by MDH, 108 (49%) were concordant by PCR. ETEC was the sixth most frequently reported bacterial enteric pathogen among a subset of CIDT-positive specimens. Sixty-nine (64%) laboratory-confirmed cases had an additional pathogen codetected with ETEC, including enteroaggregative E. coli (n = 40) and enteropathogenic E. coli (n = 39). Although travel is a risk factor for ETEC infection, only 43% of cases travelled internationally, providing evidence for ETEC as an underestimated source of domestically acquired enteric illness in the USA.


Assuntos
Escherichia coli Enterotoxigênica , Infecções por Escherichia coli/epidemiologia , Vigilância da População , Feminino , Doenças Transmitidas por Alimentos/epidemiologia , Gastroenteropatias/epidemiologia , Gastroenteropatias/microbiologia , Humanos , Masculino , Minnesota/epidemiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Estudos Retrospectivos , Estações do Ano
18.
Epidemiol Mikrobiol Imunol ; 69(2): 96-99, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32819109

RESUMO

Neonatal pneumonia is mostly bacterial and other etiology is considered less frequently. We report a case of newborn whose neonatal pneumonia has not improved, despite the aggressive ventilation regime and empiric antibiotic therapy. A special sample from the respiratory tract was collected for PCR examination. The test confirmed the presence of Trichomonas vaginalis. Antibiotic therapy was extended to include metronidazole. Targeted antibiotic therapy, which lasted for 28 days, improved the condition and the patient was discharged in a stabilized condition to home care on the 44th day of life. We demonstrate the need to consider atypical pathogens in the case of infections that do not respond to conventional therapy. The multiplex real-time PCR technique was used to detect the DNA of the pathogen. Targeted antibiotic therapy is the result of pathogen identification.


Assuntos
Pneumonia , Tricomoníase , Trichomonas vaginalis , Humanos , Recém-Nascido , Metronidazol/uso terapêutico , Reação em Cadeia da Polimerase Multiplex , Tricomoníase/diagnóstico , Tricomoníase/tratamento farmacológico , Trichomonas vaginalis/genética
19.
Niger J Clin Pract ; 23(8): 1155-1162, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32788495

RESUMO

Objective: The blaOXA resistance genes and ISAba1 were examined in 70 samples from lower respiratory tract of hospitalized patients. Materials and Methods: Of the 67 isolates obtained, almost half (46.3%) of them were from endotracheal aspirate, and most were collected from the intensive care units of the reanimation (37.3%) and internal medicine (32.8%) units. Results: Three samples from the internal medicine intensive care unit had positive cultures. Of the multidrug resistant (MDR) samples, 70 isolates (>50%) were moderately sensitive, while fewer (10%) were resistant to tigecycline. In contrast, 100% were sensitive to colistin. All strains were found to be positive for blaOXA-23-like and blaOXA-51-like genes, whereas no blaOXA-40-like and blaOXA-58-like genes were detected. The ISAba1 positivity rate was 90.0%. Pattern 5 was mainly identified among the 22 different patterns. Of note, 50% of Pattern 5 was found in the patients of the internal medicine intensive care unit, and a third was associated with ventilator-associated pneumonia. Importantly, the internal medicine unit's equipment was found to be culture positive. Conclusion: Findings obtained from this study suggest that isolates can easily spread through the hospital via isolate cross-contamination caused by health personnel. These contaminating isolates may be able to maintain their presence within the hospital for a long time.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , beta-Lactamases/genética , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/genética , Colistina/farmacologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Genes Bacterianos , Humanos , Unidades de Terapia Intensiva , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem Molecular , Reação em Cadeia da Polimerase Multiplex , Centros de Atenção Terciária
20.
Nucleic Acids Res ; 48(17): e99, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32756897

RESUMO

Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here, we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) readout. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Edição de RNA , Trypanosoma brucei brucei/genética , Fluoresceína/química , Corantes Fluorescentes/química , Genoma Mitocondrial , Reação em Cadeia da Polimerase Multiplex/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Guia/química , RNA Guia/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Uridina Trifosfato/química
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